"The figure shows a family with muscular dystrophy showing autosomal recessive inheritance. Genomic DNA was sequenced and the two mutations are shown for the affected. The corresponding control sequence for that region of the gene X is also shown.Describe the mutations in gene X in this family and its effect on the protein?"

I have gotten this question with the attached diagram. The two sequences to the left correspond to the affected female, and the two sequences to the right correspond to the unaffected male. I can see that there is a G -> N mutation in the bottom sequences and I also understand that the second mutation lies in the N's of the upper two sequences. But I do not understand what the N's tells me about the kind of mutation and how the disorder is inherited. We were told that we need to figure out why we were given two sequences and what this tells us about the inheritance. I was also told that "for a dominant disorder if there is a mutation then you will see two peaks at the same position on the gel of the same height and the computer wont be able to tell what the nucleotide is and that nucleotide is then assigned as N". How can it be a dominant disorder if the question tells us that it is an autosomal recessive disorder?

This might get you partway there. Dystrophin is a sex-linked disease carried on the X (sex) chromosome. It behaves as a recessive in females (who have two alleles for dystrophin) and as a dominant in males (who have only one dystrophin gene on their single X chromosome).

Thank you. So does that mean that the unaffected male does not carry a mutation at all (since it's dominant in males he would be affected if he had a single mutation)? But in that case where is the second mutation located (the one in the upper sequences)?

Yeah the two upper sequences are identical, but as we have been told there are 2 mutations there must be a mutation in the upper sequences as well. This must in turn mean that the unaffected male has a mutation as well.

Does anyone know how there can be 2 nucleotides located on 1 position? (As there are 2 peaks at the positions located "N", while all the other positions only have 1 nucleotide). That really confuses me.

The lower sequence shows one trace for the male but for the female there are two traces at one nucleotide position (labelled N). That looks like the allelic difference; since the female has two copies of the dystrophin gene that differ slightly, this can give rise to the two traces in the sequence (it's a single-nucleotide polymorphism). This suggests the female may be a carrier with a DMD allele and a wild-type allele for dystrophin, but the pedigree makes it look like she is expressing the DMD phenotype. That confuses me; normally if a female has a DMD allele and a wild-type allele for her dystrophin gene, she is a nonsymptomatic (or mostly nonsymptomatic) carrier.

I've not figured out what the upper sequences represent. They appear identical to me too.

jonmoulton wrote:The lower sequence shows one trace for the male but for the female there are two traces at one nucleotide position (labelled N). That looks like the allelic difference; since the female has two copies of the dystrophin gene that differ slightly, this can give rise to the two traces in the sequence (it's a single-nucleotide polymorphism). This suggests the female may be a carrier with a DMD allele and a wild-type allele for dystrophin, but the pedigree makes it look like she is expressing the DMD phenotype. That confuses me; normally if a female has a DMD allele and a wild-type allele for her dystrophin gene, she is a nonsymptomatic (or mostly nonsymptomatic) carrier.

I've not figured out what the upper sequences represent. They appear identical to me too.

I think the female has 2 mutant alleles though as the question specified that the disorder is AUTOSOMAL RECESSIVE. Therefore I am guessing that the female has 2 mutant alleles (1 mutation in the upper sequence and 1 mutation in the lower sequence), whereas the male only has 1 mutant allele (upper sequence) and 1 wildtype (lower sequence) and is therefore unaffected as it is recessive. I am not sure if muscular dystrophy actually shows this type of inheritance or if my lecturer made that up, but based on the fact that we were told it's autosomal recessive I'm guessing the woman must have 2 mutations and 2 affected alleles, could that be right?

Right - this is autosomal, therefore it is not Duchenne muscular dystrophy (DMD) and is not a mutation of the dystrophin gene. Thanks, I had missed that.

The upper sequences look identical, while the lower sequences show two different sequences for the female (due to the N position, which shows two traces) and one sequence for the male. It appears the male is a homozygote and the female is heterozygous (at least over the sequence given in the lower traces). The female might have a splice site mutation, as the sequence across the polymorphism is either ATGTGAAG or ATGGGAAG and a GT, present in only one allele, is the canonical start of an intron sequence (other factors such as splice regulatory proteins can also affect whether a potential splice site is recognized by the splicosome, so this is not a sure thing). The male shows the sequence ATGGGAAG, matching the female allele that does not contain the possible splice site. This appears to be a recessive allele.

I am still puzzled by the top trace. Is it possible that this is representing the sequence of cDNA across a splice site that is spliced alternatively, where downstream of the AAAAAAA the sequence is spliced to different sequences depending on the allele? Why, though, would the male show alternative splicing if he is homozygous?

I've clearly not worked out this problem correctly. As you learn more about this one, please post it here.